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*
Department of Infectious Diseases, Tropical Medicine, and AIDS, and
Laboratory of Experimental Internal Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands;
Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands;
ICOS Corporation, Bothell, WA; and
¶ Department of Clinical Chemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It can be anticipated that blocking of the CD14 receptor will inhibit the inflammatory response to LPS (and other bacterial Ags) and that this will be beneficial in severe sepsis. Indeed, anti-CD14 Abs, administered prophylactically or as long as 23 h after LPS injection, protected rabbits from death and renal and pulmonary injury, and prevented hypotension and leukopenia (9). In cynomolgus monkeys, pretreatment with anti-CD14 Abs derived from the same murine component 28C5 (10) as IC14 prevented LPS-induced hypotension and reduced plasma cytokine levels (11). However, at present, evidence that CD14 mediates LPS effects in humans in vivo is lacking. Therefore, in this study, we sought to determine the effect of IC14, a newly developed recombinant chimeric mAb directed against CD14, on a variety of inflammatory responses in healthy humans injected with a single dose of LPS.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sixteen healthy, male volunteers (mean age 23, range 20–33 years) were enrolled in this double-blind, randomized placebo-controlled trial. Written informed consent was obtained from all volunteers, and the study was approved by the institutional scientific and ethics committees. Medical history, physical examination, routine laboratory examination, and electrocardiogram were all normal. Tests for HIV infection and hepatitis B and C were negative. The participants did not smoke, use any medication, have any febrile illness in the month preceding the study, and never received mAb therapy before. The subjects fasted overnight before LPS administration. On the study day, blood was collected from i.v. cannulas, and LPS, IC14, or placebo was administered i.v. Eight of the volunteers received IC14, and eight were given placebo. All participants were challenged with LPS 1 h after the end of the IC14 or placebo infusion.
The study drug, IC14, was supplied by ICOS (Bothell, WA). IC14 is a
recombinant chimeric (murine/human) mAb recognizing human CD14. The
murine parent is an Ab designated 28C5 (10, 11). It is
secreted from Chinese hamster ovary cells as an
L2H2
4
Ig. Chinese hamster ovary cells were grown in bioreactors, and the
sterile harvest fluids were collected. IC14 was isolated from the
sterile harvest fluids using affinity, ion exchange, and hydrophobic
interaction chromatography steps. A dose of 1 mg/kg in a solution of
150 ml 0.9% w/v NaCl was administered i.v. during 1 h through a
0.22-µm low protein binding filter. The placebo solution consisted of
the dilution fluid and was administered in an identical manner. The
Escherichia coli endotoxin preparation used in this study,
lot G (VSP, Rockville, MD) was administered i.v. during 1 min at a dose
of 4 ng/kg 2 h after the initiation of the IC14 or placebo
infusion. The study was performed in a special research unit under
continuous supervision of physicians with emergency and resuscitation
equipment immediately available. Blood pressure, pulse rate, and
clinical symptoms were assessed every 30 min during the first 2 h
after LPS challenge and hourly thereafter. Body temperature was
measured orally. Symptoms were registered throughout the study period
using a symptom checklist and were scored by incidence and severity
(0 = absent, 1 = weak, 2 = moderate, and 3 =
severe).
Assays
Blood was obtained before the infusion of IC14 or placebo (t = -2 h), at the end of the infusion of IC14 or placebo (t = -1 h), immediately before LPS injection (t = 0 h), and at 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, and 21 h thereafter. All blood samples (except samples for flow cytometry) were centrifuged at 2000 x g for 20 min at 4°C, and plasma was stored at -20°C until assays were performed.
IC14 and soluble CD14 (sCD14) concentrations were measured in serum, and all other assays were conducted in EDTA-anticoagulated plasma. IC14 levels were measured by an enzyme immunoassay. This assay uses recombinant monomeric sCD14 immobilized on polystyrene plates to capture IC14 from serum. The captured IC14 is detected using a murine anti-human IgG4 Ab conjugated with HRP. IC14 is used as standard. The limit of detection of this assay is 6 ng/ml. SCD14 was measured by enzyme immunoassay using a murine anti-CD14 mAb (2.3 G4C) as capturing and biotin-labeled murine anti-CD14 (18E12) as detection reagent. SCD14 levels were calculated from a calibration curve, which was prepared using purified recombinant CD14. The assay does not discriminate between free sCD14 and sCD14 complexed with IC14.
Cytokines were determined by specific ELISAs according to the
instructions of the manufacturers (with detection limit): TNF-
(TNF;
1.4 pg/ml), IL-6 (0.6 pg/ml), IL-10 (1.2 pg/ml) (all obtained from the
Central Laboratory of The Netherlands Red Cross Blood Transfusion
Service, Amsterdam, The Netherlands), sTNF receptor type I (70 pg/ml),
and IL-1 receptor antagonist (IL-1RA, 204 pg/ml) (both obtained from
R&D Systems, Abingdon, U.K.). Elastase-1-antitrypsin and
lactoferrin levels were measured by specific RIA as described before
(12). Ristocetin cofactor activity of von Willebrand
factor was measured as described before (13). Soluble
E-selectin and LPS binding protein concentrations were measured by
ELISA exactly as described before (detection limits 57 and 50 pg/ml,
respectively) (14, 15). The reagents for this ELISA were
provided by W. Buurman (University of Maastricht, Maastricht, The
Netherlands). Leukocyte counts and differentials were assessed by a
STKR Coulter counter (Coulter, Bedfordshire, U.K.).
Levels of the acute phase proteins albumin,
1-antitrypsin acid glycoprotein, C-reactive protein, and
serum amyloid A were all measured with nephelometric methods on the
Behring nephelometer II (Dade Behring, Leusden, The Netherlands).
Reagents, standards, and controls were also supplied by Dade Behring.
Levels of 1-antichymotrypsin were also determined using
the Behring nephelometer II, according to the protocol of ITK
Diagnostics (Uithoorn, The Netherlands) using Dako reagents and
standard and controls from ITK Diagnostics.
Flow cytometric analysis
Saturation of CD14 receptors on circulating monocytes and granulocytes was quantitated in acid-citrate-dextrose-anticoagulated blood at -2, 0, 1, 2, 4, 6, 10, and 21 h relative to LPS injection. All blood samples were immediately placed on ice, and an equal amount of 1% paraformaldehyde solution was added. After lysis of the erythrocytes with isotonic NH4Cl solution (155 mmol/L NH4Cl, 10 mmol/L KHCO3, 0.1 mmol/L EDTA, pH 7.4), samples were centrifuged at 400 x g for 5 min. The remaining cells were washed twice in PBS with 2% v/v normal calf serum and subsequently fixed in PBS, containing 1% w/v BSA, 0.3 mmol/L EDTA, 0.01% w/v sodium azide, and 0.1% paraformaldehyde (final concentration 5 x 106 cells/ml). All subsequent procedures were performed at room temperature.
Resident IC14 was detected with FITC-labeled mouse anti-human IgG4 Ab (HP6023; Southern Biotechnology Associates, Birmingham, AL). Total CD14 was determined by adding IC14 in vitro and detecting with HP6023. All analyses were also conducted with the appropriate control Ab (murine FITC-labeled IgG3; Southern Biotechnology Associates). For each test at least 105 cells were analyzed using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). Granulocytes and monocytes were identified by forward and side-angle scatter gating. Percentage of saturation was calculated by dividing mean cell fluorescence <570 nm (MCF) of resident IC14 by the MCF of total CD14, after subtracting the MCF of the IgG3 control from each value.
Expression of CD11b and CD66 on circulating granulocytes was determined in heparinized blood obtained at -2, 0, 2, 4, 6, and 21 h. All blood samples were immediately placed on ice and processed for FACS analysis as described above. All procedures were performed at 4°C. The following Abs were used: FITC-labeled mouse anti-human CD11b and FITC-labeled mouse anti-human CD66 (both obtained from CLB, Amsterdam, The Netherlands). All FACS reagents were used in concentrations as recommended by the manufacturer, and all analyses were conducted with the appropriate control Ab (murine FITC-labeled IgG1 (Becton Dickinson, Rutherford, NJ). For each test at least 105 cells were counted. MCF of granulocytes was determined as described above. Data are presented as the difference (linear units) between MCF intensities of specifically and nonspecifically stained cells.
Phagocytosis assay
Heparinized blood was drawn immediately before and 2 h after the start of the IC14 or placebo infusion (i.e., immediately before LPS injection). Blood was kept at room temperature until use. Phagocytosis by blood monocytes and granulocytes was determined with a commercially available assay according to the instructions of the manufacturer (Phagotest; Orpegen Pharma, Heidelburg, Germany). Briefly, 100 µl of blood was added to 20 µl of FITC-labeled opsonized E. coli and incubated at 37°C for exactly 10 min. Control samples were kept on ice. Then, to suppress fluorescence by membrane-bound bacteria, 100 µl of ice-cold quenching solution (Phagotest) was added, and to stop opsonization, the cells were washed three times with ice-cold wash buffer. Thereafter, lysis buffer was added to the RBC for 20 min. Cells were washed, and DNA staining solution (propidium iodide) was added. Cells were analyzed within 1 h on a FACScan flow cytometer. Bacteria were excluded by DNA content, stained with DNA staining solution. Results are expressed as the percentage of gated cells positive or as MCF.
Calculations and statistical analysis
Values are given as mean ± SEM. Pharmacokinetic analyses were performed using standard analysis of area under the serum concentration vs time curve and area under the first moment curve. The terminal elimination half-life was calculated as ln(2)/kel, where kel is the rate constant for the terminal decline in serum IC14 concentration estimated by log-linear regression. Area under the serum concentration vs time curve and area under the first moment curve were used to estimate clearance, serum mean residence time, and terminal elimination phase volume of distribution. Differences between IC14 and placebo treatments were tested by ANOVA for repeated measures using SPSS for Windows (SPSS, Chicago, IL). Changes of parameters in time were tested using one-way ANOVA. A two-sided p value <0.05 was considered significant. Two sample comparisons were performed using the Student t test. A p value <0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Intravenous infusion of IC14 over 1 h, starting 2 h
before LPS administration, resulted in peak IC14 serum concentrations
1 h after the completion of the infusion, i.e., at the time of LPS
injection (Fig. 1
, upper
panel). Table I shows a summary of
pharmacokinetic parameters for IC14 in serum.
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Infusion of IC14 was associated with a prolonged saturation of
both monocyte and granulocyte CD14 (Fig. 1
, lower panels).
On average, no differences were found between the peak saturation
levels of either cell type. Monocyte CD14 saturation peaked 1 h
after the completion of IC14 infusion (94.9 ± 1.4%), remaining
virtually unchanged for the subsequent 6 h. At 24 h after LPS
injection, monocyte CD14 saturation still was 75.1 ± 3.5%.
Granulocyte CD14 saturation peaked 2 h after the completion of
IC14 infusion (92.1 ± 1.5%). At 24 h, granulocyte CD14
saturation was 67.3 ± 5.0%. Hence, administration of IC14 was
associated with a strong and sustained saturation of CD14 on
circulating monocytes and granulocytes.
Clinical symptoms and vital signs
LPS administration elicited flu-like symptoms such as fever,
chills, headache, nausea, vomiting, myalgia, and backache; all
volunteers were symptom-free within 24 h. Infusion of IC14 was not
associated with significant adverse effects. Administration of IC14
modestly inhibited the febrile response to LPS. Peak temperatures were
registered 4–5 h after LPS injection (placebo: 38.1 ± 0.19°C;
IC14: 37.5 ± 0.15°C; p < 0.05). In addition,
IC14 treatment attenuated all LPS-induced clinical symptoms and delayed
the time to onset of maximal severity (Table II).
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LPS administration resulted in transient increases in the plasma
levels of TNF (peak at 1.5 h: 1512 ± 463 pg/ml), IL-6 (3 h:
4583 ± 815 pg/ml), IL-10 (3 h: 288 ± 82 pg/ml), sTNF
receptor type I (2 h: 3337 ± 135 pg/ml), and IL-1RA (4 h:
33,391 ± 4,929 pg/ml) (all p < 0.05 vs time).
IC14 treatment strongly reduced the LPS-induced release of TNF (peak:
43 ± 10 pg/ml, p < 0.001), IL-6 (560 ± 137
pg/ml, p < 0.001), and IL-10 (59 ± 6 pg/ml,
p < 0.001) (Fig. 2, upper panels). IC14 only delayed LPS-induced release of the
antagonistic members of the cytokine network, sTNF receptor type I
secretion, and IL-1RA release (p < 0.05 for
both) (Fig. 2, lower panels).
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Intravenous LPS caused an initial decrease in neutrophil counts
(from 2.9 ± 0.2 to 1.4 ± 0.3 x
109/L at 1 h) followed by a neutrophilia (8
h: 17.4 ± 1.2 x 109/L,
p < 0.001 vs time). These changes were accompanied by
neutrophil activation as reflected by increases in the plasma
concentrations of the neutrophil degranulation products
elastase-1-antitrypsin complexes (from 47.4 ± 1.7
to 209.1 ± 18.9 ng/ml at 3 h, p < 0.001 vs
time) and lactoferrin (from 105.1 ± 28.2 to 487.9 ± 85.1
ng/ml at 4 h, p < 0.001 vs time). In addition,
LPS induced an up-regulation of the activation markers CD11b and CD66
on granulocytes, peaking at 6 h (MCF: CD11b from 1112 ± 147
to 4210 ± 497, p < 0.05 vs time; CD66 from
120 ± 17 to 364 ± 57, p < 0.05 vs time).
IC14 inhibited all granulocyte responses measured. IC14 blunted and
delayed the early neutropenia and reduced the subsequent neutrophilia
(Fig. 3, upper panel;
p < 0.001). The nadir in neutrophil counts occurred
only after 2 h (2.7 ± 0.4 x
109/L); maximal neutrophilia was found after
6 h (9.8 ± 0.7 x 109/L).
Moreover, IC14 attenuated neutrophil activation, as indicated by
abrogated increases in the plasma levels of
elastase-1-antitrypsin complexes (peak 133.1 ±
15.1 ng/ml, p < 0.001) and lactoferrin (peak
150.8 ± 30.3 ng/ml, p < 0.001) (Fig. 3, upper panels). Furthermore, IC14 reduced the up-regulation
of granulocyte CD11b (peak 2714 ± 340, p <
0.001) and CD66 (peak 227 ± 37, p < 0.001) (Fig. 3, lower panels).
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LPS infusion resulted in an increase of sCD14 concentrations (from
1.42 ± 0.12 to 1.92 ± 0.36 µg/ml at 24 h,
p < 0.001 vs time). IC14 administration was associated
with an earlier and more pronounced rise in IC14 levels (peak 3.46
± 0.35 µg/ml at 10 h) (Fig. 4, p < 0.001).
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LPS administration resulted in endothelial cell activation as
evidenced by increases in the plasma concentrations of von Willebrand
factor (from 87.5 ± 15.8% to 355.9 ± 39.7% at 6 h,
p < 0.001 vs time) and soluble E-selectin (from
77.6 ± 54.1 to 357.9 ± 68.8 ng/ml at 6 h)
(p < 0.001 vs time). Treatment with IC14
reduced the increases in both markers of endothelial cell activation.
Peak von Willebrand levels in subjects treated with IC14 were
240.0 ± 30.2% (p < 0.05) (Fig. 5, middle panel), peak soluble
E-selectin concentrations were 224.8 ± 51.1 ng/ml
(nonsignificant) (Fig. 5, lower panel).
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LPS elicited an acute phase response as reflected by increases in
the plasma concentrations of C-reactive protein, serum amyloid A,
1-antitrypsin, 1-antichymotrypsin,
1-acid glycoprotein, and LPS binding protein (Table III), all p < 0.05. The
release of albumin was reduced over time (p <
0.05). IC14 attenuated the increase in serum amyloid A and C-reactive
protein levels (p < 0.05) but only moderately
influenced the change in other acute phase proteins (nonsignificant,
Table III).
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CD14 is involved in the phagocytosis of bacteria by
various cell types (16, 17). Hence, blocking CD14 may have
an undesired inhibitory effect on the capacity of immunocompetent cells
to phagocytose bacteria. Therefore, we were interested to determine the
effect of IC14 infusion on the phagocytosis of FITC-labeled E.
coli by circulating monocytes and granulocytes. Infusion of
placebo did not influence phagocytosis (Table IV). Infusion of IC14 was associated with
a modest reduction in the capacity of monocytes and granulocytes to
phagocytose E. coli, when phagocytosis was expressed
as MCF (reflecting the relative number of bacteria phagocytosed per
cell; Table IV). IC14 did not change the number of monocytes or
granulocytes that phagocytosed E. coli (Table IV).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In accordance with in vitro and animal data, IC14 inhibited the proinflammatory cytokine release induced by i.v. LPS nearly completely (4, 7, 11). However, the release of the anti-inflammatory cytokine type I-sTNF receptor and IL-1RA was only delayed. Our study did not address the mechanism(s) underlying these differential effects of IC14 on agonistic and antagonistic cytokine response. We previously found that reconstituted high density lipoprotein, which, like IC14, interferes with the bioavailability of LPS, also had a more profound inhibitory effect on TNF release than on release of sTNF receptors and IL-1RA during human endotoxemia (26). These data suggest that either CD14-independent pathways are involved in sTNF receptor and IL-1RA release elicited by i.v. LPS and/or that the threshold dose at which LPS triggers these anti-inflammatory mediators is lower than the threshold LPS dose that induces TNF release. Similarly, LPS-induced clinical symptoms, which were not completely prevented by IC14, may use CD14-independent mechanisms and/or require lower LPS concentrations. In support of the former possibility are earlier findings of CD14-independent pathways for LPS and lipoarabinomannan-induced activation of monocytes in vitro (27, 28) and for LPS in mice in vivo (20).
Although monocytes are considered the main producers of cytokines
during endotoxemia, granulocytes have been implicated in the
pathogenesis of tissue injury during overwhelming sepsis by virtue of
their capacity to release potent proteinases and oxygen radicals
(29). In vitro, blocking CD14 inhibits granulocyte
responses induced by LPS (30). In rabbits, anti-CD14
treatment attenuated both the initial leukocytopenia and the subsequent
leukocytosis after i.v. administration of LPS (9). In
accordance, IC14 strongly reduced leukocyte responses elicited by i.v.
LPS in healthy humans. IC14 not only inhibited the LPS-induced early
neutropenia and later neutrophilia, but also the activation of
neutrophils, as monitored by the plasma concentrations of
elastase-1-antitrypsin complexes and lactoferrin,
indicative of neutrophil degranulation (12, 31), and the
expression of CD11b and CD66 on the surface of circulating
granulocytes.
The vascular endothelium plays a pivotal role in the inflammatory reaction to infection (18, 19). Endothelial cells lack CD14 on their surface. Instead, endothelial cells may be stimulated via LPS-sCD14 complexes or by LPS indirectly, via products of myeloid cells, i.e., cytokines (2, 10, 32). Increased levels of sCD14 have been found in sepsis patients and were associated with mortality (33, 34). In addition, sCD14 levels were increased at the site of inflammation in meningitis (35) and lung (36). In accordance with these results we found that endotoxin induced release of sCD14. However, sCD14 levels were twice as high after IC14 treatment. Because the capture ELISA for sCD14 does not discriminate between sCD14-IC14 complexes and monomeric sCD14, we consider it highly likely that the elevated sCD14 levels in IC14-treated subjects reflect sCD14-IC14 complexes. The fact that IC14 treatment was associated with an inhibition of LPS-induced endothelial cell activation as reflected by attenuated increase in the plasma levels of von Willebrand factor suggests that the sCD14-IC14 complexes were not biologically active. Furthermore, the fact that IC14 inhibits the interaction between cell surface CD14 and LPS would also imply that this Ab inhibits the interactions between sCD14 and LPS. It is difficult to directly investigate the biological activity of sCD14-IC14 complexes in blood samples obtained from our volunteers because free sCD14 can either inhibit or enhance LPS responses, depending on the cell type studied (34, 37, 38, 39), and because serum obtained from normal subjects after LPS injection by itself inhibits cytokine production (40).
The acute phase response is an important pathophysiologic phenomenon that is considered to replace the normal homeostatic mechanism with new set points that presumably contribute to defensive or adaptive capabilities (41). IC14 administration modestly attenuated the LPS-induced changes in serum amyloid A- and C-reactive protein, whereas the levels of the other acute phase proteins were not significantly influenced by IC14. In transgenic CD14-deficient mice, no change was found in the LPS-induced increase in serum amyloid A, LBP, fibrinogen, and ceruloplasmin (42). In C3H/HeJ mice, which are deficient for TLR4, the LPS-induced increase in serum amyloid A was completely abolished (42). Taken together, the induction of acute phase proteins seems to be partly CD14 independent and may be dependent on another TLR4 using signal pathway.
It should be noted that our results do not discriminate between mechanisms involving direct effects of LPS on target cells (via other CD14-independent pathways) or secondary effects of LPS-induced release of mediators occurring via CD14-independent mechanisms. The marked reduction of LPS-induced TNF release by IC14 may have contributed to the inhibition of the activation of other inflammatory pathways considering that neutralization of endogenous TNF activity inhibited these responses in endotoxemic humans (43, 44).
The results presented herein provide the first proof of principle in humans that an anti-CD14 Ab can diminish inflammatory responses induced by i.v. LPS. However, it should be noted that the role of CD14 in bacterial clearance, which, given the nature of our experiments in healthy humans, could not be investigated in this study, is not unequivocal. After infection with E. coli, CD14-deficient mice had a greatly reduced bacteremia and bacterial load in lungs compared with wild-type mice (20), whereas after infection with S. aureus bacterial load in liver, spleen, and blood were similar (45). Rabbits pretreated with anti-CD14 Abs before pulmonary inoculation with E. coli had increased bacterial outgrowth in the lung compared with control animals, but systemic inflammatory responses were prevented (46). Whether blocking CD14 on cells at the site of an infection significantly attenuates the induction of the human innate immune response to invading bacteria, thereby impairing antibacterial host defense, remains to be established. In our study, it was reassuring to find that IC14 only modestly influenced phagocytosis of E. coli by monocytes and granulocytes, indicating that in vivo CD14 may not be indispensable for an adequate ingestion of microorganisms by cells that are part of the first line of host defense. Our results indicate that blocking CD14 reduces LPS responsiveness in humans in vivo, and suggests that blocking IC14 might be of benefit in patients with evidence of systemic inflammation due to Gram-negative infection.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Annelies Verbon, Department of Internal Medicine F4-222, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.
3 T.v.d.P. is a fellow of the Royal Dutch Academy for Arts and Sciences.
4 Abbreviations used in this paper: TLR, Toll-like receptor; s, soluble; IL-1RA, IL-1 receptor antagonist; MCF, mean cell fluorescence <570 nm.
Received for publication September 11, 2000. Accepted for publication December 20, 2000.
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 K. A. Hintz, A. J. Rassias, K. Wardwell, M. L. Moss, P. M. Morganelli, P. A. Pioli, A. L. Givan, P. K. Wallace, M. P. Yeager, and P. M. Guyre Endotoxin induces rapid metalloproteinase-mediated shedding followed by up-regulation of the monocyte hemoglobin scavenger receptor CD163 J. Leukoc. Biol., October 1, 2002; 72(4): 711 - 717. [Abstract] [Full Text] [PDF]
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I. Ishida, H. Kubo, S. Suzuki, T. Suzuki, S. Akashi, K. Inoue, S. Maeda, H. Kikuchi, H. Sasaki, and T. Kondo Hypoxia Diminishes Toll-Like Receptor 4 Expression Through Reactive Oxygen Species Generated by Mitochondria in Endothelial Cells J. Immunol., August 15, 2002; 169(4): 2069 - 2075. [Abstract] [Full Text] [PDF]
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) or placebo (•) was given i.v.
from -2 to -1 h relative to endotoxin injection (4 ng/kg;
t = 0 h).
